Stem Cell Epigenetics News

January 2017

TRIM28 Controls a Gene Regulatory Network Based on Endogenous Retroviruses in Human Neural Progenitor Cells
Endogenous retroviruses (ERVs), which make up 8% of the human genome, have been proposed to participate in the control of gene regulatory networks. In this study, we find a region- and developmental stage-specific expression pattern of ERVs in the developing human brain, which is linked to a transcriptional network based on ERVs. We demonstrate that almost 10,000, primarily primate-specific, ERVs act as docking platforms for the co-repressor protein TRIM28 in human neural progenitor cells, which results in the establishment of local heterochromatin. Thereby, TRIM28 represses ERVs and consequently regulates the expression of neighboring genes. These results uncover a gene regulatory network based on ERVs that participates in control of gene expression of protein-coding transcripts important for brain development.
Brattås et al. (2016) Cell Reports. doi:10.1016/j.celrep.2016.12.010.Abstract.

The mammal-specific Pdx1 Area II enhancer has multiple essential functions in early endocrine cell specification and postnatal β-cell maturation
The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5′ conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (−2139 to −1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo. Pdx1ΔAREAII/− mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions.
Yang et al. (2017) Development. doi:10.1242/dev.143123.Abstract.Active Motif products relevant to this paper:RNA Subcellular Isolation Kit

TET-mediated DNA demethylation controls gastrulation by regulating Lefty–Nodal signalling
Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined. Here a collaboration of researchers from China and the US show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty-Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, their results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty-Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.
Dai et al. (2016) Nature. doi:10.1038/nature20095.Abstract.

Isoform switch of TET1 regulates DNA demethylation and mouse development
The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, the authors found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.
Zhang et al. (2016) Mol Cell. doi:10.1016/j.molcel.2016.Abstract.Active Motif products relevant to this paper:Tet1 Antibody

November 2016

Cooperative action between SALL4A and TET proteins in stepwise oxidation of 5-methylcytosine
TET family enzymes successively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to eventual demethylation. 5-hmC and TET enzymes occupy distinct chromatin regions, suggesting unknown mechanisms controlling the fate of 5-hmC within diverse chromatin environments. Here, researchers at the Chinese Academy of Sciences in Beijing report that SALL4A preferentially associates with 5-hmC in vitro and occupies enhancers in mouse embryonic stem cells in a largely TET1-dependent manner. Although most 5-hmC at SALL4A peaks undergoes further oxidation, this process is abrogated upon deletion of Sall4 gene, with a concomitant reduction of TET2 at these regions. Thus, SALL4A facilitates further oxidation of 5-hmC at its binding sites, which requires its 5-hmC-binding activity and TET2, supporting a collaborative action between SALL4A and TET proteins in regulating stepwise oxidation of 5-mC at enhancers. This study identifies SALL4A as a 5-hmC binder, which facilitates 5-hmC oxidation by stabilizing TET2 association, thereby fine-tuning expression profiles of developmental genes in mouse embryonic stem cells.
Xiong et al. (2016) Mol. Cell. doi:10.1016/j.molcel.2016.10.013Abstract.

Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex
Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, scientists in the UK show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. The authors propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.
Miller et al. (2016) Development. doi:10.1242/dev.139113.Abstract.

October 2016

Histone H3 globular domain acetylation identifies a new class of enhancers
Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here the authors show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, they find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.
Pradeepa et al. (2016) Nat Genet. doi:10.1038/ng.3550.Abstract.

Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network
Pluripotent mouse embryonic stem cells maintain their identity throughout virtually infinite cell divisions. This phenomenon, referred to as self-renewal, depends on a network of sequence-specific transcription factors (TFs) and requires daughter cells to accurately reproduce the gene expression pattern of the mother. However, dramatic chromosomal changes take place in mitosis, generally leading to the eviction of TFs from chromatin. Here, scientists at the Pasteur Institute in Paris, France report that Esrrb, a major pluripotency TF, remains bound to key regulatory regions during mitosis. They show that mitotic Esrrb binding is highly dynamic, driven by specific recognition of its DNA-binding motif and is associated with early transcriptional activation of target genes after completion of mitosis. These results indicate that Esrrb may act as a mitotic bookmarking factor, opening another perspective to molecularly understand the role of sequence-specific TFs in the epigenetic control of self-renewal, pluripotency and genome reprogramming.
Festuccia et al. (2016) , Nat Cell Biol. doi:10.1038/ncb3418.Abstract.

A pre-neoplastic epigenetic field defect in HCV-infected liver at transcription factor binding sites and polycomb targets
The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumours is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. The authors studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events. They find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on their samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas. Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression. The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically reversible epigenetic field defect in HCV-infected liver.
Wijetunga et al. (2016) Oncogene. doi:10.1038/onc.2016.340.Abstract.Active Motif products relevant to this paper:DNA Methylation Services

September 2016

MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells
The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. The authors of this recent Nature Scientific Reports publication have developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. As such, a new RNA tool, miR-switch, was synthesized as a live-cell reporter mRNA for miR-302a activity. This new tool can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and prevent teratoma formation in an in vivo tumorigenicity assay. This system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells and holds great potential for investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.
Parr et al. (2016) Nat Scientific Reports. DOI: 10.1038/srep32532Abstract.

Cellular Metabolism and Induced Pluripotency
Cellular metabolism involves a set of complex and highly coordinated life-sustaining biochemical reactions that convert or use energy to maintain the living state of cells. Cellular metabolism has long been considered as a consequence, rather than a driver, of cell-fate changes—a view that has recently been challenged by its intrinsic links to epigenetic modifications of chromatin during development, disease progression, and cellular reprograming. In this Review, the authors summarize what we know about metabolic pathways characteristic of pluripotent stem cells (PSCs) and discuss metabolic reprograming to induced pluripotency and the modeling of metabolic diseases with iPSCs.
Wu et al. (2016) Cell. http://dx.doi.org/10.1016/j.cell.2016.08.008Abstract.Active Motif products relevant to this paper: LightSwitch™ Luciferace Assay System

DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells
Mutations in the epigenetic modifiers DNMT3A and TET2 non-randomly co-occur in lymphoma and leukemia despite their epistasis in the methylation-hydroxymethylation pathway. Using Dnmt3a and Tet2 double-knockout mice in which the development of malignancy is accelerated, researchers at the Baylor College of Medicine in Houston, Texas show that the double-knockout methylome reflects regions of independent, competitive and cooperative activity. Expression of lineage-specific transcription factors, including the erythroid regulators Klf1 and Epor, is upregulated in double-knockout hematopoietic stem cells (HSCs). DNMT3A and TET2 both repress Klf1, suggesting a model of cooperative inhibition by epigenetic modifiers. These data demonstrate a dual role for TET2 in promoting and inhibiting HSC differentiation, the loss of which, along with DNMT3A, obstructs differentiation, leading to transformation.
Zhang et al. (2016) , Nat Genet. doi:10.1038/ng.3610.Abstract.

Zfp281 coordinates opposing functions of Tet1 and Tet2 in pluripotent states
Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, the authors performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. They identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. The study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states.
Fidalgo et al. (2016) , Cell Stem Cell. doi:10.1016/j.stem.2016.01.007.Abstract.

Regulation of the DNA methylation landscape in human somatic cell reprogramming by the miR-29 family
Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, scientist found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Their global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. These findings uncover an important miRNA-based approach to generate clinically robust iPSCs.
Hysolli et al. (2016) Stem Cell Reports. doi:10.1016/j.stemcr.2016.05.014.Abstract.Active Motif products relevant to this paper: DNA Methylation (5-hmC, 5-caC, 5-fC, etc.) Antibodies